This project will elucidate the structure and mechanism of the Group II intron, a catalytic RNA molecule (or ribozyme) which remains enzymologically and structurally uncharacterized. Group II intron self- splicing is central to the metabolism of higher plants and it shares mechanistic features with the eukaryotic pre-mRNA splicing apparatus. For this reason, it may constitute a model for the chemical and structural elements involved in all eukaryotic RNA splicing. Because of its similarities to our own forms of RNA processing, the Group II intron may provide insight into the remnants of RNA catalysis which remains important in higher organisms. The active site and substrate specificity of the Group II intron appear different from those of other ribozymes, so its characterization will push back the limits of known RNA reactivity and increase the base of knowledge required for application of ribozymes in gene therapy. The first objective of this study is to compare the reactivity of known self-splicing Group II introns to learn which one is most kinetically efficient and to understand how differences in Group II morphology affect their reactivity. Efficiency of the introns in two- step and single-step reactions will be compared by monitoring rates of exon ligation and cleavage from 32p-labeled RNA transcripts. Based on this study, a particular Group II intron will become the focus of further investigation. The second objective is to determine the location of tertiary interactions between catalytically essential Domains 1 and 5 of the Group II intron. These interactions are unusual forms of RNA-RNA contact potentially analogous to the ribose 2;-OH--base contacts I identified in the Tetrahymena ribozyme. Techniques of in-vitro selection and RNA footprinting will aid in the identification of critical 2'- OH, phosphate and base functionalities which are required for proper tertiary structure formation . The third objective is to convert the Group II self-splicing RNA from a unimolecular species to a multiple-turnover ribozyme suitable for detailed enzymological analysis of the first step of splicing. It is the first step of Group II splicing which is potentially the most unusual. The multiple-turnover ribozyme will be created by fragmenting the intron into catalytically essential Domains 1 and 5, by transcription of a Domain 5-Domain 6 nucleophilic cofactor and synthesis of a ribozyme "substrate" analogous to 5'exon-intron boundary sequences. The multicomponent ribozyme will be analyzed under conditions which will yield the individual rate and binding constants descriptive of the active site and catalytic mechanism. The kinetic framework will then be used to test models for Group II intron reactivity through mutagenesis and functional group substitution on the individual components. The framework will also facilitate a fourth objective: to establish analogies between subdomains of the Group II intron and small nuclear RNAs (snRNAs) of the eukaryotic splicing apparatus (the spliceosome). U and U2 snRNAs will be inserted in place of Domain 5 and a portion of Domain 6 in reaction of the multiple turnover construct. Exchange in catalytic function would provide firm evidence for RNA catalysis within the ribonucleoprotein spliceosome.